Purpose: Endoplasmic reticulum (ER) and mitochondrial stress are independently associated with corneal endothelial cell (CEnC) loss in many corneal diseases, including Fuchs’ endothelial corneal dystrophy (FECD). However, the role of ER stress in mitochondrial dysfunction contributing to CEnC apoptosis is unknown. The purpose of this study is to explore the crosstalk between ER and mitochondrial stress in CEnC. Methods: Human corneal endothelial cell line (HCEnC-21T) and human corneal endothelial tissues were treated with ER stressor tunicamycin. ER stress-reducing chemical 4-phenyl butyric acid (4-PBA) was used in HCEnC-21T after tunicamycin. Fuchs’ corneal endothelial cell line (F35T) was used to determine differential activation of ER stress with respect to HCEnC-21T at the baseline. ER stress, mitochondrial-mediated intrinsic apoptotic, mitochondrial fission, and fusion proteins were determined using immunoblotting and immunohistochemistry. Mitochondrial bioenergetics were assessed by mitochondrial membrane potential (MMP) loss and ATP production at 48 hours after tunicamycin. Mitochondria dynamics (shape, area, perimeter) were also analyzed at 24 hours using transmission electron microscopy. Results: Treatment of HCEnC-21T cell line with tunicamycin activated three ER stress pathways (PERK-eIF2α-CHOP, IRE1α-XBP1, and ATF6), reduced cell viability, upregulated mitochondrial-mediated intrinsic apoptotic molecules (cleaved caspase 9, caspase 3, PARP, Bax, cytochrome C), downregulated anti-apoptotic Bcl-2 protein, initiated mitochondrial dysfunction by loss of MMP and lowering of ATP production, and caused mitochondrial swelling and fragmentation with increased expression of mitochondrial fission proteins (Fis1 and p-Drp1). Fuchs’ CEnC (F35T) cell line also showed activation of the ER stress-related proteins (p-eIF2α, GRP78, CHOP, XBP1) compared to HCEnC-21T at the baseline. The 4-PBA ameliorated cell loss and reduced cleaved caspase 3 and 9, thereby rescuing tunicamycin-induced cell death but not mitochondrial bioenergetics in HCEnC-21T cell line. Conclusions: Tunicamycin-induced ER stress disrupts mitochondrial bioenegetics, dynamics and contributes to the loss of CEnC viability. This novel study highlights the importance of ER-mitochondria crosstalk and its contribution to CEnCs apoptosis, seen in many corneal diseases, including FECD.

Endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and mitophagy are known to contribute independently to corneal endothelial cell (CEnC) apoptosis in Fuchs’ endothelial corneal dystrophy (FECD). However, the role of a specific ER stress pathway (PERK-ATF4-CHOP) in regulating these events is unknown. This study aims to investigate the role of ATF4 in regulating mitochondrial dysfunction and mitophagy, which ultimately leads to CEnC apoptosis. Human corneal endothelial cell line (21T), Fuchs’ corneal endothelial cell line (F35T), and primary human corneal endothelial cells were treated with ER stressor tunicamycin (Tun). ATF4 siRNA was used to knock down ATF4 in 21T cell line and primary corneal endothelial cells. Mitophagy and apoptotic proteins were analyzed using Western blotting. ATF4^+/− and ATF4 ^+/+ mice were irradiated with UVA to assess ER stress and corneal endothelial apoptosis. F35T cell line had significantly increased expression of the ER stress pathway as well as caspase-mediated apoptotic molecules compared to 21T at baseline, which further increased after Tun treatment. F35T cells exhibited significantly decreased mitochondrial ATP and MMP, and increased mitochondrial fragmentation, which was further exacerbated after Tun. F35T cells also demonstrated inhibition of mitophagy, similar to 21T, after treatment with tunicamycin, despite the upregulation of mitophagy initiators. ATF4 knockdown attenuated ER and mitochondrial stress proteins, rescued mitochondrial membrane potential (MMP) loss, downregulated mitochondrial fragmentation, activated mitophagy, and prevented CEnC death under chronic ER stress. ATF4^+/− mice had increased CE numbers, with improved cellular morphology and decreased ER stress marker CHOP expression, compared to ATF4^+/+ mice post-UVA. Pro-apoptotic ATF4 induction following ER stress disrupts mitochondrial function, leading to mitophagy inhibition and CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD.

Fuchs endothelial corneal dystrophy (FECD) is a progressive and debilitating disorder of the corneal endothelium (CE) that affects approximately 4% of individuals over the age of 40. Despite the burden of the disease, the pathogenesis of FECD remains poorly understood, and treatment options are limited, highlighting the need for deeper investigation into its underlying molecular mechanisms. Over the past decade, studies have indicated independent contributions of endoplasmic reticulum (ER) and mitochondrial stress to the pathogenesis of FECD. However, there are limited studies suggesting ER-mitochondria crosstalk in FECD. Recently, our lab established the role of chronic ER stress in inducing mitochondrial dysfunction for corneal endothelial cells (CEnCs), indicating the existence of ER-mitochondria crosstalk in FECD. This paper aims to provide a comprehensive overview of the current understanding of how ER and mitochondrial stress contribute to FECD pathogenesis. The paper also reviews the literature on the mechanisms of ER-mitochondria crosstalk in other diseases relevant to FECD.

PURPOSE: Endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and mitophagy are known to contribute independently to corneal endothelial cell (CEnC) apoptosis in Fuchs’ endothelial corneal dystrophy (FECD). However, the role of a specific ER stress pathway (PERK-ATF4-CHOP) in regulating these events is unknown. This study aims to investigate the role of ATF4 in regulating mitochondrial dysfunction and mitophagy, which ultimately leads to CEnC apoptosis in FECD. METHODS: Human corneal endothelial cell line (21T), Fuchs’ corneal endothelial cell line (F35T), and primary human corneal endothelial cells were treated with ER stressor tunicamycin (Tun). ATF4 siRNA was used to knock down ATF4 in 21T cell line and primary corneal endothelial cells. Mitophagy and apoptotic proteins were analyzed using Western blotting. ATF4 ^+/- and ATF4 ^+/+ mice were irradiated with UVA to assess ER stress and corneal endothelial apoptosis. RESULTS: F35T cell line had significantly increased expression of the ER stress pathway as well as caspase-mediated apoptotic molecules compared to 21T at baseline, which further increased after tunicamycin treatment. F35T cells exhibited significantly decreased ATP and MMP, and increased mitochondrial fragmentation, which was further exacerbated after Tunicamycin. F35T cell line also demonstrated inhibition of mitophagy, similar to 21T, after treatment with Tunicamycin, despite the upregulation of mitophagy initiators. ATF4 knockdown attenuated ER and mitochondrial stress proteins, rescued mitochondrial membrane potential (MMP) loss, downregulated mitochondrial fragmentation, activated mitophagy, and prevented cell death under chronic ER stress. ATF4 ^+/- mice had increased CE numbers, with improved cellular morphology and decreased ER stress CHOP expression, compared to ATF4+/+ mice post-UVA. CONCLUSIONS: Pro-apoptotic ATF4 induction following ER stress disrupts mitochondrial function, leading to mitophagy inhibition and CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD.

Purpose: Endoplasmic reticulum (ER) stress-mediated cell death contributes to many ocular diseases, including Fuchs corneal endothelial dystrophy (FECD). Natural compounds possess significant pharmacological activities necessary for vital cell signaling pathways and are considered protective in many ocular diseases such as diabetes, retinopathy, and glaucoma. However, there are limited studies examining the role of natural compounds in reducing sustained ER stress for improving corneal endothelial cell (CEnCs) survival. The purpose of this study is to identify readily available natural compounds to improve the viability of CEnCs under ER stress. Methods: Human corneal endothelial cells (21T-HCEnC) were cultured and seeded in 384-well plates, pre-treated with 502 compounds from a natural product library (Enzo Screen-Well Natural Product library), and then subjected to ER stressor tunicamycin (1µg/mL) for 24 hours. Cell viability was measured using the Cell-Titer-Glo assay, and 74 compounds improved viability past the set threshold (≥ 30% increase in viability) and were selected for further dose-response testing. Further dose-response testing was performed using untreated F35T corneal endothelial cells, plated onto 384-well plates, and treated with a range of 6 log doses of each compound, from 10 pg/mL up to 1 μg/mL. After 24 hours of treatment, cell viability was measured again using the Cell-Titer Glo assay. Of the 4 natural compounds that improved viability past the set threshold at the lowest dose tested (0.00001 µg/mL), veratrine was analyzed further via western blotting, ranging from 10 pg/mL to 0.01 μg/mL. Apoptotic and unfolded protein response markers were also measured via western blotting. Results: Of 502 compounds each administered at 0.01 µg/mL, we identified 74 compounds exhibiting 30% or more protection of 21T HCEnC cell viability in response to tunicamycin-induced stress. Of the 74 compounds that underwent further dose-response testing, 4 compounds improved cell viability by 30% or more at the lowest dose of compound tested compared to viability of F35T cells at baseline. Cevadine, or veratrine, was chosen for further analysis due to its function as a sodium channel agonist and its interaction with Na ⁺ /K ⁺ ATPase channels. Pretreatment with veratrine 0.01 µg/mL, along with tunicamycin, downregulated expression levels of cleaved caspase 9, PARP, cytochrome C, Bax, and cleaved caspase 3. Western blots of FECD cell line under tunicamycin-induced ER stress, pretreated with veratrine 0.01 µg/mL, also showed reduced markers from all three ER unfolded protein response pathways. Conclusions: This study provided insight into the role of natural compounds in regulating ER stress-mediated CEnC death associated with many corneal endothelial-associated diseases. Veratrine was found to have a protective effect on ER-stressed CEnCs, as indicated by reduced apoptotic signals and ER stress markers following veratrine treatment, suggesting a possible mechanism of action via mediating ER stress signals. Future studies are needed to clarify the mechanism and utility of veratrine in FECD.

Cells respond to metabolic and environmental challenges through the integrated stress response (ISR), a cellular process that maintains homeostasis under diverse stressors. ATF4 is a key player in this ISR, as it is activated via the PERK–eIF2α–ATF4 pathway. ATF4 induction can elicit adaptive responses, including the regulation of genes involved in metabolism and autophagy, to maintain homeostasis. However, ATF4 activation can also induce apoptosis, leading to a wide spectrum of diseases, including metabolic, neurologic, and ocular pathologies. This duality reflects the highly context-dependent nature of ATF4 signaling. This review aims to synthesize the role of ATF4 in metabolic dysfunction, neurodegenerative diseases, and ocular pathology; the mechanisms underlying its protective versus pathologic effects; and future directions to refine ATF4’s potential as a clinical therapeutic target across different diseases.